Immunogenic peptides from the HPV E7 protein

ABSTRACT

The invention provides immunogenic peptides from the HPV type 16 E7 protein that comprise overlapping class I restricted T cell epitopes. Also disclosed are methods of administering DNA molecules encoding these peptides to a host mammal.

This application claims priority from currently pending U.S. Ser. No.60/061,657 (herein incorporated by reference), which was filed Oct. 9,1997.

BACKGROUND OF THE INVENTION

This invention relates to treatment of human papilloma virus (HPV)infection.

Papilloma viruses are non-enveloped DNA viruses with a double strandedcircular genome of approximately 8,000 bp. Over 75 types of humanpapilloma viruses (HPV) have been typed at the DNA level, and these canbe broadly grouped into families on the basis of their tissue tropism.

Histologic, molecular, and epidemiologic evidence have implicated someHPV strains in cervical dysplasia and cervical cancer. Many studiessupport the view that most moderate and severe cervical intraepithelialneoplasias (CIN) contain HPV DNA which is exclusively detected in thehistologically abnormal epithelium of these lesions. Persistentinfection with HPV is believed to be the predominant risk factor fordevelopment of cervical carcinoma. HPV DNA is readily found in episomalform within cells exhibiting a cytopathic effect, while the HPV DNA isfound integrated within the chromosomes of cells associated with mosthigh grade precancerous lesions and cancer. Approximately 23 HPV typesare commonly found in anogenital screening programs, but only 10-15 areassociated with progressive disease. Type 16 is the type most commonlyfound in cervical cancer tissue.

Papillomaviruses contain nine open reading frames. HPV genes withtransforming properties have been mapped to open reading frames E6 andE7. Substantial biochemical work has demonstrated that the HPV E6protein inactivates the protein p53, whereas the E7 protein interfereswith retinoblastoma (Rb) protein function. Since p53 and Rb aretumor-suppressor proteins which function as cell division inhibitors,their inactivation by E6 and E7 leads the cell to enter into S phase ofthe cell cycle. Expression of E6 and E7 is sufficient to immortalizesome primary cell lines, and blocking E6 or E7 function has been shownto reverse the transformed state.

SUMMARY OF THE INVENTION

The invention is based on the discovery that a 13 amino acid peptidefrom the HPV strain 16 E7 protein that contains overlapping class I HLAbinding, T cell epitopes can induce a CTL response in an animal.Accordingly, the invention includes an immunogenic peptide having withinits sequence multiple class I MHC-binding epitopes from a humanpapillomavirus (HPV) protein, and which has a length of less than 19amino acids and includes the sequence of Leu Met Gly Thr Leu Gly Ile ValCys Pro Ile Cys (SEQ ID NO:16) (hereinafter “immunogenic peptide”). Theimmunogenic peptide can optionally include sequences in addition tothose derived from the E7 protein.

The immunogenic peptide can have the sequence of Leu Leu Met Gly Thr LeuGly Ile Val Cys Pro Ile Cys (SEQ ID NO:3) or Xaa Leu Met Gly Thr Leu GlyIle Val Cys Pro Ile Cys, Xaa being Met, Ala, Ser, Arg, Lys, Gly, Gln,Asp, or Glu (SEQ ID NO:19), e.g., Ala Leu Met Gly Thr Leu Gly Ile ValCys Pro Ile Cys (SEQ ID NO:4).

The invention also includes the peptides Thr Leu Gly Ile Val Cys Pro Ile(SEQ ID NO:20) and Gly Thr Leu Gly Ile Val Cys Pro Ile (SEQ ID NO:21),as well as Xaa Thr Leu Gly Ile Val Cys Pro Ile (SEQ ID NO:27) and GlyThr Leu Gly Leu Gly Ile Val Cys Pro Ile (SEQ ID NO:28), Xaa being Met,Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu.

In addition, all of the peptides discussed herein may include additionalamino acids to facilitate expression, e.g., an amino terminal methionineto facilitate translation.

The invention also includes a polypeptide having the sequence of a firstpeptide linked to a second peptide by a peptide bond. The first peptide(which can be at the carboxy terminus or the amino terminus of thesecond peptide, so long as it functions in that site) is a peptide whichcontrols intracellular trafficking of a peptide to which it is attached,and the second peptide is the immunogenic peptide described above. Thepolypeptide may optionally be modified to introduce an amino acidsubstitution at the junction between the first and second peptides topromote cleavage of the first and second peptides by a signal peptidase.

The trafficking peptides can be any recognized signal sequence, e.g. asignal sequence from the adenovirus E3 protein. A preferred traffickingpeptide is the signal peptide of HLA-DRα, Met Ala Ile Ser Gly Val ProVal Leu Gly Phe Phe Ile Ile Ala Val Leu Met Ser Ala Gln Glu Ser Trp Ala(SEQ ID NO:18).

The invention in addition includes a therapeutic composition containingthe immunogenic peptide described above, and a pharmaceuticallyacceptable carrier. The polypeptide can optionally be formulated in amicroparticle, a liposome or an immune-stimulating complex (ISCOM)(which may contain saponin alone as the active ingredient), or any othervehicle suitable for delivering into subjects the immunogenic peptidesof the invention. When a microparticle is used, it preferably has apolymeric matrix that is a copolymer such as poly-lactic-co-glycolicacid (PLGA).

An immune response (e.g., a cellular immune response, including an MHCclass I-mediated or class II-mediated immune response) in a mammal canbe elicited by administering the immunogenic peptide to a mammal, e.g.,a human, non-human primate, dog, cat, rabbit, cow, mouse, rat, guineapig, or hamster, that has an MHC molecule that binds to the immunogenicpeptide. The immunogenic peptide can be administered as part of amicroparticle, liposome, or ISCOM, or in solution.

Another way to administer the peptide utilizes a nucleic acid, e.g., anexpression vector, comprising a coding sequence encoding the immunogenicpeptide. The nucleic acid can optionally encode a signal sequence linkedto the immunogenic peptide, as described above. When the nucleic acidencodes such a signal sequence, it is preferred that it encodes thesignal sequence from HLA-DRα (SEQ ID NO:18). In such a case, theimmunogenic peptide can have the sequence, for example, of SEQ ID NO:4or SEQ ID NO:3. Preferably, the nucleic acid does not include sequencesfrom a viral genome that would render the nucleic acid infectious, anddoes not encode an intact E7 protein.

The nucleic acid described above can be included in a plasmid,optionally provided in a microparticle that also includes a polymericmatrix. In preferred embodiments, the polymeric matrix consistsessentially of a copolymer of PLGA. The microparticle preferably has adiameter of, e.g., 0.02 to 20 microns, or less than about 11 microns. Aplurality of microparticles preferably has diameter of, e.g., 0.02 to 20microns, or less than about 11 microns

Also within the invention is a cell containing the plasmid of theinvention. The cell can, e.g., be a B cell or other antigen presentingcell (APC). The cell may be cultured or otherwise maintained underconditions permitting expression of the peptide from the plasmidencoding it.

The nucleic acid and plasmid of the invention are useful in a method ofinducing an immune response in a mammal, e.g., a human, by administeringthe above-described plasmid to the mammal, e.g., as “naked DNA”. Themammal may be at risk for, or suffer from, HPV infection, cervicaldysplasia, and/or cervical cancer. The nucleic acids and plasmids of theinvention can also be incorporated into microparticles, liposomes,ISCOMS, or any other suitable delivery vehicle as described above.

The invention further includes a plasmid having a sequence essentiallyidentical to that of pBIOTOPE_(HPV) (SEQ ID NO:7), or a microparticleconsisting essentially of a PLGA polymeric matrix and the pBIOTOPE_(HPV)plasmid, as well as methods of inducing an immune response in a mammalby administering either the plasmid alone, or the plasmid incorporatedinto such a microparticle, to the mammal.

By a “substantially pure polypeptide” is meant a polypeptide which isseparated from those components (proteins and other naturally-occurringorganic molecules) which naturally accompany it. Typically, thepolypeptide is substantially pure when it constitutes at least 60%, byweight, of the protein in the preparation. Preferably, the protein inthe preparation consists of at least 75%, more preferably at least 90%,and most preferably at least 99%, by weight, of an immunogenic peptide.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The preferred methods andmaterials for practicing the invention are described below, althoughother methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention.All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present application, including definitions, willcontrol. The materials, methods, and examples are illustrative only andnot intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of the pBIOTOPE_(HPV) plasmid.

FIG. 2 is a graph showing the results of subjecting cells to triplecolor flow cytometry staining for CD8, CD16, and interferon-gamma.

FIG. 3 is a graph showing CTL lysis of an HLA-A2⁺, HPV16⁺ cell line withT cells from an HLA-A2⁺ donor stimulated with an influenza peptide(-▪-), the A2.1 peptide (-•-), or the A2.4-C peptide (-▴-).

FIG. 4 is a graph showing CTL lysis of an HLA-A2⁺, HPV16⁺ cell line withT cells from a second HLA-A2⁺ donor stimulated with an influenza peptide(-▪-), the A2.1 peptide (-•-), or the A2.4 peptide (-▴-).

DETAILED DESCRIPTION

The peptides disclosed herein, and the nucleic acids encoding thepeptides, can be used to elicit an immune response against the HPV E7protein. The peptides were identified in part based on their bindingaffinity with the MHC class I HLA-A2 allele. Thus, the immune responseelicited by these peptides is likely to be class I-mediated but may alsoinvolve class II mediated responses, B cell responses, or NK cellresponses. The immune response can thus involve, e.g., cells expressingMHC class I molecules or cells expressing MHC class II molecules. Theimmune response can also include immune cells such as macrophages,polymorphonuclear monocytes (PMN), natural killer cells, and B cells.

Five immunogenic peptides derived from the HPV type 16 E7 protein areshown in Table I. Peptide A2.¼, Leu Leu Met Gly Thr Leu Gly Ile Val CysPro Ile Cys (SEQ ID NO:3), corresponds to amino acids 82-94 in the HPVType 16 E7 protein and includes the overlapping sequences of peptidesA2.1, Leu Leu Met Gly Thr Leu Gly Ile Val (SEQ ID NO:1), A2.4, Thr LeuGly Ile Val Cys Pro Ile Cys (SEQ ID NO:2) A2.4-C, Thr Leu Gly Ile ValCys Pro Ile (SEQ ID NO:20), and 2.5, Gly Thr Leu Gly Ile Val Cys Pro Ile(SEQ ID NO:21). Thus, peptide A2.¼ has at least four overlappingepitopes potentially recognized by class I MHC restricted T cells.

TABLE I Amino acid sequences of conserved, class I-MHC binding, TCRbinding HPV strain 16 E7 peptides A2.1 LLMGTLGIV (SEQ ID NO:1) A2.4TLGIVCPIC (SEQ ID NO:2) A2.1/4 LLMGTLGIVCPIC (SEQ ID NO:3) A2.4-CTLGIVCPI (SEQ ID NO:20) A2.5 GTLGIVCPI (SEQ ID NO:21)

A peptide of the invention may optionally include one having the aminoacids SQK added to the carboxy terminus of the A2.¼ peptide sequence(“the extended peptide”). Processing of the extended peptide cangenerate the peptide IVCPICSQK (SEQ ID NO:22), which has been reportedas binding the MHC class I molecules HLA-A3 and HLA-A11 (Kast et al., J.Immunol. 152:3904-11, 1994). This region of the HPV E7 protein hasseveral peptides that can be processed into MHC binding peptides.Additional extensions to the amino or carboxy terminus of the A2.¼peptide may further increase the number of peptides that can begenerated from this region of the E7 protein.

The peptides of the invention can be linked to a trafficking sequencethat directs the peptides to a desired intracellular compartment. Atrafficking sequence is an amino acid sequence which functions tocontrol intracellular trafficking (directed movement from organelle toorganelle or to the cell surface) of a polypeptide to which it isattached. Such trafficking sequences might traffic the polypeptide toER, a lysosome, or an endosome, and include signal peptides (the aminoterminal sequences which direct proteins into the ER duringtranslation), ER retention peptides such as KDEL (SEQ ID NO:23), andlysosome-targeting peptides such as KFERQ (SEQ ID NO:28), QREFK (SEQ IDNO:30), and other pentapeptides having Q flanked on one side by fourresidues selected from K, R, D, E, F, I, V, and L.

Short amino acid sequences can act as signals to target proteins tospecific intracellular compartments. For example, hydrophobic signalpeptides are found at the amino terminus of proteins destined for theER, while the sequence KFERQ (SEQ ID NO:29) (and other closely relatedsequences) is known to target intracellular polypeptides to lysosomes,while other sequences target polypeptides to endosomes.

One such trafficking sequence is the HLA-DRα leader sequence, Met AlaIle Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val Leu Met Ser AlaGln Glu Ser Trp Ala (SEQ ID NO:18). The signal peptide may include onlya portion (e.g., at least ten amino acid residues) of the specified 25residue sequence, provided that portion is sufficient to causetrafficking of the polypeptide to the ER.

In some cases it is desirable to modify the portion of the peptidespanning the trafficking sequence and the sequence encoding the HPV E7antigenic peptide to facilitate processing, i.e., cleavage, by thesignal peptidase. Recognition sequences for signal peptides aredescribed in Von Heijne, NAR 14:4683, 1986.

Standard techniques can be used to construct a DNA encoding theantigenic peptide (see, e.g., the techniques described in WO 94/04171).The construct may include additional sequences for enhancing expressionin human cells, e.g., appropriate promoters, RNA stabilization sequences5′ and 3′ to the coding sequence, introns (which can be placed at anylocation 5′ or 3′ within encoded sequence), and poly(A) addition sites,as well as an origin of replication and selectable markers enabling theconstructs to replicate and be selected for in prokaryotic and/oreukaryotic hosts.

An example of a DNA sequence encoding an immunogenic HPV E7 antigen isthe BIOTOPE_(HPV) construct (SEQ ID NO:7), which is shown schematicallyin FIG. 1. This plasmid contains a minigene (SEQ ID NO:5) at positions3290-3413. The minigene encodes the HLA-DRα trafficking peptide linkedto 12 residues of the A2.¼ peptide. In the peptide encoded by theminigene, an alanine has been substituted for the amino terminal leucinein the A2.¼ peptide in order to facilitate cleaving of the traffickingpeptide from the immunogenic peptide by a signal peptidase. TheBIOTOPE_(HPV) construct also carries the immediate early promoter ofhuman cytomegalovirus (CMV) at positions 2619-3315, and RNAstabilization sequences (RST) derived from the Xenopus laevis β-globingene flanking the minigene (positions 3219-3279 and 3426-3624). Tomaximize export from the nucleus, the pre-mRNA expressed from theplasmid contains a chimeric intron between the coding sequence of theminigene and the SV40 polyadenylation site. The intron can also functionif located between the promoter and the coding region.

Once in the cytoplasm of the cell, the mRNA transcribed from theminigene is translated to produce a 40 amino acid hybrid peptide. Thefirst two amino acids are methionine and aspartic acid (derived fromvector sequences), and the next 38 amino acids correspond to Met Ala IleSer Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val Leu Met Ser Ala GlnGlu Ser Trp Ala Ala Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQID NO:6). The amino-terminal 25 amino acids of the 38-residue portionare identical in sequence to the non-polymorphic HLA-DRα chain geneleader sequence (SEQ ID NO:18). The last 13 amino acids have thesequence Ala Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ IDNO:4), which is the A2.¼ peptide described above, but with an alanineresidue substituted for the amino terminal leucine residue.

Also within the plasmid is a kanamycin resistance gene (positions519-1313), which is driven by the SV40 early promoter (positions131-484) and which has a thymidine kinase (TK) polyadenylation site(positions 1314-1758). The kanamycin resistance gene and accompanyingregulatory sequences are for selection purposes only and can be removedfrom the plasmid if selection is not required or desired.

Once expressed in a cell, the encoded peptide can be processed into oneof several HLA MHC class I binding epitopes. At least some of these areincluded in Table 1. These peptides can bind the HLA-A2 allele and mayalso bind other alleles, such as HLA-A1, HLA-A3, HLA-A11, HLA-A24. TheMHC molecule, upon binding to the peptide, can activate a T cellresponse. MHC class II binding peptides may also be generated from theencoded peptide. These peptides would be expected to activate T helpercells or CTL upon presentation by the MHC class II expressing cells.Other receptors may also bind the encoded peptide or its processedfragments to activate immune cells such as NK or B cells. These cellsmay also be activated by cytokines elicited in response to the peptidesof the invention.

The peptides and nucleic acids of the invention can be used as vaccinesprophylactically or therapeutically in subjects known to be infected byHPV, suspected of being infected by HPV, or likely to become infected byHPV. Other suitable subjects include those displaying symptoms of, orlikely to develop, HPV-associated conditions. The immunogenic peptides,and nucleic acids encoding these peptides, can be used as vaccines inpreventing or treating conditions associated with infections of HPVstrain 16, e.g., bowenoid papulosis, anal dysplasia, respiratory orconjunctival papillomas, cervical dysplasia, cervical cancer, vulvalcancer, or prostate cancer. They can also be used to treat conditionsassociated with other HPV strains, especially those associated with HPVstrains 18, 45, 6, 11, 35 and 31, which have regions of homology to thepeptide of SEQ ID NO:3. These conditions include, e.g., exophyticcondyloma (HPV strains 6 and 11), flat condyloma, especially of thecervix (HPV strains 6, 11, 16, 18, and 31), giant condyloma (HPV strains6 and 11), cervical cancer (HPV strains 18, 31, and 33, in addition toHPV strain 16), respiratory and conjunctival papillomas (HPV 6 and 11),and infection with genital-tract HPVs (HPV 6, 11, and 16).

The immunogenic peptides or nucleic acids encoding the peptides canadministered alone or in combination with other therapies known in theart, e.g., chemotherapeutic regimens, radiation, and surgery, to treatHPV infections, or diseases associated with HPV infections. In addition,the peptides and nucleic acids of the invention can be administered incombination with other treatments designed to enhance immune responses,e.g., by co-administration with adjuvants or cytokines (or nucleic acidsencoding cytokines) as is well known in the art.

The peptides or nucleic acids of the invention can also be used inmanufacture of a medicament for the prevention or treatment of HPVinfection, or conditions associated with HPV infection.

Delivery of Immunuogenic Peptides and Nucleic Acids Encoding ImmunogenicPeptides

The delivery systems of the invention may be used to deliver, intoappropriate cells, peptides, or DNA constructs which express peptides,intended to stimulate an immune response against HPV. An advantage ofDNA delivery is that the antigenic peptide is produced inside the targetcell itself, where the interaction with a class I or class II MHCmolecule to which the immunogenic peptide binds is kinetically favored.This is in contrast to standard vaccine protocols which do notspecifically direct antigenic peptides to MHC molecules. In addition,the immune response directly stimulated by DNA vaccines of the inventionis likely to be limited to a T cell mediated response, in contrast tostandard vaccine protocols which result in a more generalized immuneresponse, although it is possible that an antibody response may beindirectly induced when cells bearing viral particles are killed, or byother mechanisms.

The immunogenic peptides, or nucleic acids encoding the peptides, can beadministered using standard methods, e.g., those described in Donnellyet al., J. Imm. Methods 176:145, 1994, and Vitiello et al., J. Clin.Invest. 95:341, 1995. Peptides and nucleic acids of the invention can beinjected into subjects in any manner known in the art, e.g.,intramuscularly, intravenously, intraarterially, intradermally,intraperitoneally, intranasally, intravaginally, intrarectally orsubcutaneously, or they can be introduced into the gastrointestinaltract, the mucosa, or the respiratory tract, e.g., by inhalation of asolution or powder containing the microparticles. Administration can belocal (e.g., at the cervix or other site of infection) or systemic.

The immunogenic peptides and nucleic acids encoding immunogenic peptidescan be delivered in a pharmaceutically acceptable carrier such assaline, lipids, liposomes, microspheres, nanospheres, as colloidalsuspensions, or as powders. They can be naked or associated or complexedwith delivery vehicles and delivered using delivery systems known in theart, such as lipids, liposomes, microparticles, gold, nanoparticles,polymers, condensing agents, polysaccharides, polyamino acids,dendrimers, saponins, adsorption enhancing materials, or fatty acids.

It is expected that a dosage of approximately 0.1 to 100 μmoles of thepolypeptide, or of about 1 to 200 μg of DNA, would be administered perkg of body weight per dose. Where the patient is an adult human,vaccination regimens can include, e.g., intramuscular, intravenous,oral, or subcutaneous administrations of 10-1000 μg of pBIOTOPE_(HPV)DNA when delivered in a microparticle, or of about 100-1000 μg of nakedpBIOTOPE_(HPV) DNA delivered intramuscularly or intradermally, repeated3-6 times. Of course, as is well known in the medical arts, dosage forany given patient depends upon many factors, including the patient'ssize, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Determination of optimaldosage is well within the abilities of a pharmacologist of ordinaryskill.

Other standard delivery methods, e,g, biolistic transfer, or ex vivotreatment, can also be used. In ex vivo treatment, e.g., antigenpresenting cells (APCs), dendritic cells, peripheral blood mononuclearcells, or bone marrow cells, can be obtained from a patient or anappropriate donor and activated ex vivo with the immunogeniccompositions, and then returned to the patient.

Microparticle Delivery of Synthetic Immunogenic Peptides or PlasmidsEncoding Immunogenic Peptides

Microparticles, including those described in U.S. Pat. No. 5,783,567,can be used as vehicles for delivering macromolecules such as DNA, RNA,or polypeptides into cells. They contain macromolecules embedded in apolymeric matrix or enclosed in a shell of polymer. Microparticles actto maintain the integrity of the macromolecule e.g., by maintainingenclosed DNA in a nondegraded state. Microparticles can also be used forpulsed delivery of the macromolecule, and for delivery at a specificsite or to a specific cell or target cell population.

The polymeric matrix can be a biodegradable co-polymer such aspoly-lactic-co-glycolic acid, starch, gelatin, or chitin. Microparticlescan be used in particular to maximize delivery of DNA molecules into asubject's phagocytotic cells. Alternatively, the microparticles can beinjected or implanted in a tissue, where they form a deposit. As thedeposit breaks down, the nucleic acid is released gradually over timeand taken up by neighboring cells (including APCs) as free DNA.

Liposomal Delivery of Synthetic Immunogenic Peptides or PlasmidsEncoding Immunogenic Peptides

The immunogenic peptides of the invention can be administered intosubjects via lipids, dendrimers, or liposomes using techniques that arewell known in the art. For example, liposomes carrying immunogenicpolypeptides or nucleic acids encoding immunogenic peptides are known toelicit CTL responses in vivo (Reddy et al., J. Immunol. 148:1585, 1992;Collins et al., J. Immunol. 148:3336-3341, 1992; Fries et al., Proc.Natl. Acad. Sci. USA 89:358, 1992; Nabel et al., Proc. Nat. Acad. Sci.(USA) 89:5157, 1992).

Delivery of Synthetic Immunogenic Peptides or Plasmids EncodingImmunogenic Peptides Using Saponin

The peptides and nucleic acids of the invention can be administered byusing Immune Stimulating Complexes (ISCOMS), which are negativelycharged cage-like structures of 30-40 nm in size formed spontaneously onmixing cholesterol and Quil A (saponin), or saponin alone. The peptidesand nucleic acids of the invention can be co-administered with theISCOMS, or can be administered separately.

Protective immunity has been generated in a variety of experimentalmodels of infection, including toxoplasmosis and Epstein-Barrvirus-induced tumors, using ISCOMS as the delivery vehicle for antigens(Mowat et al., Immunology Today 12:383-385, 1991). Doses of antigen aslow as 1 μg encapsulated in ISCOMS have been found to produce class Imediated CTL responses, where either purified intact HIV-1-IIIB gp 160envelope glycoprotein or influenza hemagglutinin is the antigen(Takahashi et al., Nature 344:873-875, 1990).

Measuring Responses of the Immune System and of HPV Virus Infections tothe Immunogenic Peptides or Nucleic Acids Encoding the ImmunogenicPeptides

The ability of immunogenic peptides, or nucleic acids encoding the same,to elicit an immune response can be assayed by using methods formeasuring immune responses that are well known in the art. For example,the generation of cytotoxic T cells can be demonstrated in a standard⁵¹Cr release assay, by measuring intracellular cytokine expression, orby using MHC tetramers. Standard assays, such as ELISA or ELISPOT, canalso be used to measure cytokine profiles attributable to T cellactivation. T cell proliferation can also be measured using assays suchas ³H-thymidine uptake and other assays known in the art. B cellresponses can be measured using art recognized assays such as ELISA.

Other methodologies, e.g., digital imaging, cytologic, colposcopic andhistological evaluations, can also be used to evaluate the effects ofimmunogenic peptides, and of nucleic acids encoding the immunogenicpeptides, on papilloma virus-associated lesions, or on papilloma viruslevels generally.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES

As described in the Examples below, experimental models were chosen todemonstrate the generation of vigorous CTL responses to plasmidsencoding the immunogenic peptides of the invention, e.g.,pBIOTOPE_(HPV).

Initial screening of HPV peptide sequences was performed by assessingbinding affinity to the human class I HLA-A2 molecule. This was done bymeasuring the changes in circular dichroism (CD) as the receptor/ligandcomplex “melted”. Examples of this type of screening are shown inExample 1. Of particular interest in Example 1 was the hybrid peptideA2.¼, which contains at least two known epitopes.

Using a murine transgenic model, plasmids containing minigenes encodingthese peptides were evaluated for their ability to generate HLA-A2restricted CTLS (Examples 2 and 3). CTL activity, as measured usinghuman target cells labeled with HPV peptides, was significantlyincreased over control targets for both the plasmids encoding A2.4 andA2.¼, including the pA2.4 plasmid delivered in a PLGA microparticle.

Example 1 Peptides Derived From HPV Strain 16 E7 Protein Bind PurifiedHLA-A*0201 with High Affinity

To determine if peptides A2.1 (SEQ ID NO:l), A2.2 (SEQ ID NO:17), A2.4(SEQ ID NO:2) A2.¼ (SEQ ID NO:3), and A2.¼, bind with biologicalaffinity to the human class I molecule HLA-A2 (for the peptides A2.1,A2.2, A2.4 and A2.¼) or HLA-A3 (for the A2.¼-SQK peptide), recombinantHLA-A2 or HLA-A3 was produced in E. coli and refolded in the presence ofthe HPV-derived peptides and purified human β₂-microglobulin. Theresulting peptide-HLA complexes were then further purified by HPLC. Todetermine the precise thermokinetic interaction energy between receptorand ligand, each complex was “melted” while its structure was monitoredby circular dichroism. The temperature required to “melt” the complex isan accurate indication of the affinity between receptor and ligand.

The results of the binding studies are shown in Table II.

Table II. Peptides binding HLA-A molecules

TABLE II NAME Amino Acid Sequence IC₅₀¤ Tm♦ A2.1 SEQ ID NO:1 8 47.8 A2.2SEQ ID NO:17 49 52.5 A2.4 SEQ ID NO:2 153 41.5 A2.1/4 SEQ ID NO:3 ND41.0 A2.1/4SQK ND 47.8 ¤IC₅₀ represents tha amount (nM) of peptiderequired for 50% inhibition of binding of a radiolabeled standardpeptide to HLA-A*0201 or HLA-A*0301 measured in a molecular bindingassay. ♦Values represent the temperature in degrees Celsius at which 50%of the refolded complexes are melted. HLA-A2 and HLA-A3 will not refoldin the absence of a peptide ligand of sufficient affinity.

Of particular interest is a hybrid peptide A2.¼, which contains at leasttwo known overlapping epitopes, A2.1 and A2.4, each of which ispresented by HLA-A2 positive human cervical tumor cells expressing theHPV 16 E7 protein (Ressing et al., J. Immunology 154:5934, 1995). Of thepeptides studied, A2.4 is predicted to be the most capable of elicitingcross reactive immune responses between HPV strains. Moreover, thehybrid peptide generates both the A2.1 and A2.4 peptides; administrationof pBIOTOPE_(HPV) to mice was found to generate T cell responses to bothimmunogenic peptides.

Example 2 Induction of HPV-specific CTL in HLA-transgenic Mice Immunizedwith Intramuscular Injections of a Plasmid Encoding the HPV Strain 16Derived A2.4 Peptide

To demonstrate that a plasmid encoding the A2.4 peptide (SEQ ID NO:2)produced HPV peptides in vivo and that CTL to these peptides weregenerated, a transgenic animal model was employed. The HLA-A2/K^(b)mouse line produces a hybrid MHC class I molecule. In this hybrid, thepeptide binding domains (α1 and α2) are derived from the human class Imolecule HLA-A*0201, whereas the domain (α3) which interacts with theCD8 co-receptor on CTLs is derived from the murine class I moleculeK^(b). The resulting animal is capable of responding to immunogens whichcontain HLA-A2 restricted epitopes and of generating murine CTLs thatrecognize human target cells expressing HLA-A2 (Vitiello et al., J. Exp.Med. 173:1007, 1991).

6-8 week old HLA-A2/K^(b) females were immunized with either a plasmidencoding the A2.4 peptide having the amino terminal leucine replacedwith an alanine residue, or with a null vector. Injections wereperformed with 50 μg of plasmid DNA injected as “naked DNA” (that is,with no liposome, microparticle, or other carrier) into each anteriortibialis muscle. A booster immunization was performed 14 days after thefirst immunization, and a second booster immunization was performed 14days after the first boost. Ten days following the third immunization,splenocytes were harvested and stimulated in vitro with syngeneiclipopolysaccharide (LPS) blasts which had been incubated with thesynthetic A2.4 peptide. After 4 days of co-culture, CTL activity wasmeasured on human targets labeled with HPV peptides (Table III).

TABLE III Lysis of Human Cells Labeled with HPV-derived Peptides byMurine CTL from HLA-Transgenic Mice Immunized with Plasmid Encoding anA2.4 peptide. IMMUNOGEN % LYSIS OF TARGET CELLS* pVA2.4 28.7 ± 0.7*Vector  6.8 ± 2.9* *Data are reported as the mean lysis values at 100:1effector to target ratio. Error is reported as the standard deviation; p= 0.05 by Students t-test.

Mice immunized with a plasmid encoding the A2.4 peptide generate CTLthat lyse human targets expressing HLA-A2 and the appropriate HPVpeptide. This response is significantly greater than that achieved byimmunization with null vector DNA alone.

Example 3 Plasmid DNA Encoding the A2.¼ Peptide Delivered to Mice inPLGA Microparticles Elicits CTL Responses

6-8 week old HLA-A2/K^(b) females were immunized intraperitoneally onetime with 2-5 μg of PLGA microparticles containing plasmidpBIOTOPE_(HPV). Seven days following the immunization, splenocytes wereharvested and in vitro stimulated with IL-2. After 2 days, CTL activitywas measured on human targets labeled with HPV peptides (HPV(+)), orlacking HPV peptide (HPV(−)), at an E:T ratio of 50:1 (Table IV).

TABLE IV Lysis of Human Cells Labeled with HPV- derived Peptides byMurine Splenocytes from HLA- Transgenic Mice Immunized with PLGAMicroparticles Containing pBIOTOPE_(HPV) % LYSIS OF TARGET CELLSIMMUNOGEN HPV(+) HPV(−) pBIOTOPE_(HPV) 17.4 ± 2.8* 3.9 ± 4.2* Data arereported as the mean lysis values from three individual measurements.*Error is reported as the standard deviation; p value <0.05 asdetermined by the Students t-test.

Thus, mice immunized with PLGA microparticles containing pBIOTOPE_(HPV)generate CTL that lyse human targets expressing HLA-A2 and A2.¼ peptide.

Example 4 Synthetic Peptides Derived from HPV Type 16 Activate Human CTL

Peripheral blood mononuclear cells (PBMC) from an HLA-A2⁺ donor werecultured in vitro for two rounds of stimulation in the presence of 300units of IL-2 and peptide A2.¼ (LLMGTLGIVCPIC) (SEQ ID NO:3) or animmunodominant peptide having the amino acid sequence GILGFVFTL (SEQ IDNO:24) from influenza virus, which was used as a positive control.

Seven days after the second stimulation, each culture was subdividedinto two subgroups. One subgroup of each culture was stimulated for anadditional 7 hours with the respective peptide (“the third peptidestimulation”), while the other subgroups were cultured without thepeptide. All samples were pretreated with brefeldin A to preventcytokine secretion. The cells were then subjected to triple color flowcytometry staining for CD8, CD16, and interferon-γ.

The results of the experiments are shown in FIG. 2. For cells treatedwith the A2.¼ peptide, 28% of the cells subjected to the third peptidestimulation stained positive for interferon-γ, compared to 1.7% of thecells that did not receive a final stimulation. The percentage of CD8⁺cells in cells receiving a third stimulation with peptide was 14.4%,while 19.2% of the cells which did not received a third stimulation wereCD8⁺. Overall, 3.1% of the PBMC receiving a final pulse of the A2.¼peptide were activated CTL, i.e., were CD8⁺ CD16⁻ IFN-γ⁺, compared to0.5% of the cells receiving no final pulse of HPV-derived peptide.

For cells treated with the influenza peptide, 11.5% of the cellsreceiving a third stimulation with the influenza peptide were positivefor interferon γ, compared to 1.7% of the cells that did not receive athird stimulation. For cells cultured with influenza peptide, 11.5% ofthe cells given a final pulse of influenza peptide were activated,compared to 1.49% of cells which were not given a final pulse ofinfluenza.

FIGS. 3 and 4 demonstrate that CTL specific for the A2.1 or A2.4-Cpeptides can recognize and lyse HPV 16-infected cells. FIG. 3 showsCTL-mediated lysis of an HLA-A2⁺, HPV-16⁺ transformed line (Caski) by Tcell populations exposed to peptide A2.1, A2.4-C, or a peptide derivedfrom influenza virus (“Flu”). Effector/target (E/T) ratios ranging from25 to 1.5 were used. The peptide A2.4-C was highly effective at inducinglysis, with nearly 35% release detected at an E/T ratio of 25:1. TheA2.1 peptide was less effective, but nevertheless caused much higherpercentages of lysis at E/T ratios of 25:1 and 12:1 than did theinfluenza peptide.

Results with PBL isolated from a second HLA-A2⁺ individual and subjectedto two rounds of stimulation with peptide A2.1, peptide A2.4, or theinfluenza peptide (“Flu”) are shown in FIG. 4. Both the A2.1 and A2.4peptides induced higher levels of lysis than did the influenza peptide.

These observations demonstrate that the A2.¼ peptide, or peptidesderived thereform, can activate and expand PBL from humans, and thatthese peptides can cause CTL-mediated lysis of target cells transformedwith HPV16.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, that the foregoingdescription is intended to illustrate and not limit the scope of theappended claims. Other aspects, advantages, and modifications are withinthe scope of the following claims.

33 9 amino acids amino acid linear peptide unknown 1 Leu Leu Met Gly ThrLeu Gly Ile Val 1 5 9 amino acids amino acid linear peptide unknown 2Thr Leu Gly Ile Val Cys Pro Ile Cys 1 5 13 amino acids amino acid linearpeptide unknown 3 Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys 15 10 13 amino acids amino acid linear peptide unknown 4 Ala Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys 1 5 10 117 base pairs nucleic aciddouble linear DNA unknown Coding Sequence 1...114 5 ATG GCC ATA AGT GGAGTC CCT GTG CTA GGA TTT TTC ATC ATA GCT GTG 48 Met Ala Ile Ser Gly ValPro Val Leu Gly Phe Phe Ile Ile Ala Val 1 5 10 15 CTG ATG AGC GCT CAGGAA TCA TGG GCT GCC CTG ATG GGC ACC CTG GGC 96 Leu Met Ser Ala Gln GluSer Trp Ala Ala Leu Met Gly Thr Leu Gly 20 25 30 ATC GTG TGC CCC ATC TGCTGA 117 Ile Val Cys Pro Ile Cys 35 38 amino acids amino acid linearprotein internal unknown 6 Met Ala Ile Ser Gly Val Pro Val Leu Gly PhePhe Ile Ile Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu Ser Trp Ala AlaLeu Met Gly Thr Leu Gly 20 25 30 Ile Val Cys Pro Ile Cys 35 4665 basepairs nucleic acid double linear DNA unknown 7 GCACTTTTCG GGGAAATGTGCGCGGAACCC CTATTTGTTT ATTTTTCTAA ATACATTCAA 60 ATATGTATCC GCTCATGAGACAATAACCCT GATAAATGCT TCAATAATAT TGAAAAAGGA 120 AGAGTCCTGA GGCGGAAAGAACCAGCTGTG GAATGTGTGT CAGTTAGGGT GTGGAAAGTC 180 CCCAGGCTCC CCAGCAGGCAGAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG 240 GTGTGGAAAG TCCCCAGGCTCCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA 300 GTCAGCAACC ATAGTCCCGCCCCTAACTCC GCCCATCCCG CCCCTAACTC CGCCCAGTTC 360 CGCCCATTCT CCGCCCCATGGCTGACTAAT TTTTTTTATT TATGCAGAGG CCGAGGCCGC 420 CTCGGCCTCT GAGCTATTCCAGAAGTAGTG AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG 480 CAAAGATCGA TCAAGAGACAGGATGAGGAT CGTTTCGCAT GATTGAACAA GATGGATTGC 540 ACGCAGGTTC TCCGGCCGCTTGGGTGGAGA GGCTATTCGG CTATGACTGG GCACAACAGA 600 CAATCGGCTG CTCTGATGCCGCCGTGTTCC GGCTGTCAGC GCAGGGGCGC CCGGTTCTTT 660 TTGTCAAGAC CGACCTGTCCGGTGCCCTGA ATGAACTGCA AGACGAGGCA GCGCGGCTAT 720 CGTGGCTGGC CACGACGGGCGTTCCTTGCG CAGCTGTGCT CGACGTTGTC ACTGAAGCGG 780 GAAGGGACTG GCTGCTATTGGGCGAAGTGC CGGGGCAGGA TCTCCTGTCA TCTCACCTTG 840 CTCCTGCCGA GAAAGTATCCATCATGGCTG ATGCAATGCG GCGGCTGCAT ACGCTTGATC 900 CGGCTACCTG CCCATTCGACCACCAAGCGA AACATCGCAT CGAGCGAGCA CGTACTCGGA 960 TGGAAGCCGG TCTTGTCGATCAGGATGATC TGGACGAAGA GCATCAGGGG CTCGCGCCAG 1020 CCGAACTGTT CGCCAGGCTCAAGGCGAGCA TGCCCGACGG CGAGGATCTC GTCGTGACCC 1080 ATGGCGATGC CTGCTTGCCGAATATCATGG TGGAAAATGG CCGCTTTTCT GGATTCATCG 1140 ACTGTGGCCG GCTGGGTGTGGCGGACCGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA 1200 TTGCTGAAGA GCTTGGCGGCGAATGGGCTG ACCGCTTCCT CGTGCTTTAC GGTATCGCCG 1260 CTCCCGATTC GCAGCGCATCGCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAGCGGGAC 1320 TCTGGGGTTC GAAATGACCGACCAAGCGAC GCCCAACCTG CCATCACGAG ATTTCGATTC 1380 CACCGCCGCC TTCTATGAAAGGTTGGGCTT CGGAATCGTT TTCCGGGACG CCGGCTGGAT 1440 GATCCTCCAG CGCGGGGATCTCATGCTGGA GTTCTTCGCC CACCCTAGGG GGAGGCTAAC 1500 TGAAACACGG AAGGAGACAATACCGGAAGG AACCCGCGCT ATGACGGCAA TAAAAAGACA 1560 GAATAAAACG CACGGTGTTGGGTCGTTTGT TCATAAACGC GGGGTTCGGT CCCAGGGCTG 1620 GCACTCTGTC GATACCCCACCGAGACCCCA TTGGGGCCAA TACGCCCGCG TTTCTTCCTT 1680 TTCCCCACCC CACCCCCCAAGTTCGGGTGA AGGCCCAGGG CTCGCAGCCA ACGTCGGGGC 1740 GGCAGGCCCT GCCATAGCCTCAGGTTACTC ATATATACTT TAGATTGATT TAAAACTTCA 1800 TTTTTAATTT AAAAGGATCTAGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC 1860 TTAACGTGAG TTTTCGTTCCACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC 1920 TTGAGATCCT TTTTTTCTGCGCGTAATCTG CTGCTTGCAA ACAAAAAAAC CACCGCTACC 1980 AGCGGTGGTT TGTTTGCCGGATCAAGAGCT ACCAACTCTT TTTCCGAAGG TAACTGGCTT 2040 CAGCAGAGCG CAGATACCAAATACTGTTCT TCTAGTGTAG CCGTAGTTAG GCCACCACTT 2100 CAAGAACTCT GTAGCACCGCCTACATACCT CGCTCTGCTA ATCCTGTTAC CAGTGGCTGC 2160 TGCCAGTGGC GATAAGTCGTGTCTTACCGG GTTGGACTCA AGACGATAGT TACCGGATAA 2220 GGCGCAGCGG TCGGGCTGAACGGGGGGTTC GTGCACACAG CCCAGCTTGG AGCGAACGAC 2280 CTACACCGAA CTGAGATACCTACAGCGTGA GCTATGAGAA AGCGCCACGC TTCCCGAAGG 2340 GAGAAAGGCG GACAGGTATCCGGTAAGCGG CAGGGTCGGA ACAGGAGAGC GCACGAGGGA 2400 GCTTCCAGGG GGAAACGCCTGGTATCTTTA TAGTCCTGTC GGGTTTCGCC ACCTCTGACT 2460 TGAGCGTCGA TTTTTGTGATGCTCGTCAGG GGGGCGGAGC CTATGGAAAA ACGCCAGCAA 2520 CGCGGCCTTT TTACGGTTCCTGGCCTTTTG CTGGCCTTTT GCTCACATGT TCTTTCCTGC 2580 GTTATCCCCT GATTCTGTGGATAACCGTAT TACCGCCATG CATTAGTTAT TAATAGTAAT 2640 CAATTACGGG GTCATTAGTTCATAGCCCAT ATATGGAGTT CCGCGTTACA TAACTTACGG 2700 TAAATGGCCC GCCTGGCTGACCGCCCAACG ACCCCCGCCC ATTGACGTCA ATAATGACGT 2760 ATGTTCCCAT AGTAACGCCAATAGGGACTT TCCATTGACG TCAATGGGTG GAGTATTTAC 2820 GGTAAACTGC CCACTTGGCAGTACATCAAG TGTATCATAT GCCAAGTACG CCCCCTATTG 2880 ACGTCAATGA CGGTAAATGGCCCGCCTGGC ATTATGCCCA GTACATGACC TTATGGGACT 2940 TTCCTACTTG GCAGTACATCTACGTATTAG TCATCGCTAT TACCATGGTG ATGCGGTTTT 3000 GGCAGTACAT CAATGGGCGTGGATAGCGGT TTGACTCACG GGGATTTCCA AGTCTCCACC 3060 CCATTGACGT CAATGGGAGTTTGTTTTGGC ACCAAAATCA ACGGGACTTT CCAAAATGTC 3120 GTAACAACTC CGCCCCATTGACGCAAATGG GCGGTAGGCG TGTACGGTGG GAGGTCTATA 3180 TAAGCAGAGC TGGTTTAGTGAACCGTCAGA TCCGCTAGAG CTTGCTTGTT CTTTTTGCAG 3240 AAGCTCAGAA TAAACGCTCAACTTTGGCAG ATCCGCGGCT CGAGCCACCA TGGACATGGC 3300 CATAAGTGGA GTCCCTGTGCTAGGATTTTT CATCATAGCT GTGCTGATGA GCGCTCAGGA 3360 ATCATGGGCT GCCCTGATGGGCACCCTGGG CATCGTGTGC CCCATCTGCT GAGCTCCTGG 3420 AATTCGGATC TGGTTACCACTAAACCAGCC TCAAGAACAC CCGAATGGAG TCTCTAAGCT 3480 ACATAATACC AACTTACACTTTACAAAATG TTGTCCCCCA AAATGTAGCC ATTCGTATCT 3540 GCTCCTAATA AAAAGAAAGTTTCTTCACAT TCTAAAAAAA AAAAAAAAAA AAAAAAAAAA 3600 AAAAAACCCC CCCCCCCCCCCCCCATCGAT TTTCCACCCG GGTGGGGTAC CAGGTAAGTG 3660 TACCCAATTC GCCCTATAGTGAGTCGTATT ACAATTCACT GGCCGTCGTT TTACAACGTC 3720 GTGACTGGGA AAACCCTGGCGTTACCCAAA TTAATCGCCT TGCAGCACAT CCCCCTTTCG 3780 CCAGCTGGCG TAATAGCGAAGAGGCCCGCA CCGATCGCCC TTCCCAACAG TTGCGCAGCC 3840 TGAATGGCGA ATGGAGATCCAATTTTTAAG TGTATAATGT GTTAAACTAC TGATTCTAAT 3900 TGTTTGTGTA TTTTAGATTCACAGTCCCAA GGCTCATTTC AGGCCCCTCA GTCCTCACAG 3960 TCTGTTCATG ATCATAATCAGCCATACCAC ATTTGTAGAG GTTTTACTTG CTTTAAAAAA 4020 CCTCCCACAC CTCCCCCTGAACCTGAAACA TAAAATGAAT GCAATTGTTG TTGTTAACTT 4080 GTTTATTGCA GCTTATAATGGTTACAAATA AAGCAATAGC ATCACAAATT TCACAAATAA 4140 AGCATTTTTT TCACTGCATTCTAGTTGTGG TTTGTCCAAA CTCATCAATG TATCTTAACG 4200 CGTAAATTGT AAGCGTTAATATTTTGTTAA AATTCGCGTT AAATTTTTGT TAAATCAGCT 4260 CATTTTTTAA CCAATAGGCCGAAATCGGCA AAATCCCTTA TAAATCAAAA GAATAGACCG 4320 AGATAGGGTT GAGTGTTGTTCCAGTTTGGA ACAAGAGTCC ACTATTAAAG AACGTGGACT 4380 CCAACGTCAA AGGGCGAAAAACCGTCTATC AGGGCGATGG CCCACTACGT GAACCATCAC 4440 CCTAATCAAG TTTTTTGGGGTCGAGGTGCC GTAAAGCACT AAATCGGAAC CCTAAAGGGA 4500 GCCCCCGATT TAGAGCTTGACGGGGAAAGC CGGCGAACGT GGCGAGAAAG GAAGGGAAGA 4560 AAGCGAAAGG AGCGGGCGCTAGGGCGCTGG CAAGTGTAGC GGTCACGCTG CGCGTAACCA 4620 CCACACCCGC CGCGCTTAATGCGCCGCTAC AGGGCGCGTC AGGTG 4665 27 base pairs nucleic acid singlelinear DNA unknown 8 GGCGTCGACA TGGCCATAAG TGGAGTC 27 27 base pairsnucleic acid single linear DNA unknown 9 GAAGCTGGCA GCCCATGATT CCTGAGC27 27 base pairs nucleic acid single linear DNA unknown 10 TCATGGGCTGCCAGCTTCGA GGCCCAG 27 27 base pairs nucleic acid single linear DNAunknown 11 CGGGAATTCT TAGGCCTTGT CCACGGC 27 61 base pairs nucleic acidsingle linear DNA unknown 12 ATCAGCGCTC AGGAATCATG GGCTGCCCTG GGCATCGTGTGCCCCATCTG CTGAGCTCGA 60 G 61 24 base pairs nucleic acid single linearDNA unknown 13 GGGGATCCGA ATTCCTCGAG CTCA 24 70 base pairs nucleic acidsingle linear DNA unknown 14 ATCAGCGCTC AGGAATCATG GGCTCTGATG GGCACCCTGGGCATCGTGTG CCCCATCTGC 60 TGAGCTCGAG 70 24 base pairs nucleic acid singlelinear DNA unknown 15 GGGGATCCGA ATTCCTCGAG CTCA 24 12 amino acids aminoacid linear peptide unknown 16 Leu Met Gly Thr Leu Gly Ile Val Cys ProIle Cys 1 5 10 9 amino acids amino acid linear peptide unknown 17 TyrMet Leu Asp Leu Gln Pro Glu Thr 1 5 25 amino acids amino acid linearpeptide unknown 18 Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe IleIle Ala Val 1 5 10 15 Leu Met Ser Ala Gln Glu Ser Trp Ala 20 25 13 aminoacids amino acid linear peptide unknown Other 1...1 where Xaa atposition 1 is Met, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu 19 Xaa LeuMet Gly Thr Leu Gly Ile Val Cys Pro Ile Cys 1 5 10 8 amino acids aminoacid linear peptide unknown 20 Thr Leu Gly Ile Val Cys Pro Ile 1 5 9amino acids amino acid linear peptide unknown 21 Gly Thr Leu Gly Ile ValCys Pro Ile 1 5 9 amino acids amino acid linear peptide unknown 22 IleVal Cys Pro Ile Cys Ser Gln Lys 1 5 4 amino acids amino acid linearpeptide unknown 23 Lys Asp Glu Leu 1 9 amino acids amino acid linearpeptide unknown 24 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 16 aminoacids amino acid linear peptide unknown 25 Leu Leu Met Gly Thr Leu GlyIle Val Cys Pro Ile Cys Ser Gln Lys 1 5 10 15 8 amino acids amino acidlinear peptide unknown 26 Met Gly Ile Val Cys Pro Ile Cys 1 5 9 aminoacids amino acid linear peptide unknown Other 1...1 where Xaa atposition 1 is Met, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu 27 Xaa ThrLeu Gly Ile Val Cys Pro Ile 1 5 11 amino acids amino acid linear peptideunknown 28 Gly Thr Leu Gly Leu Gly Ile Val Cys Pro Ile 1 5 10 5 aminoacids amino acid linear peptide unknown 29 Lys Phe Glu Arg Gln 1 5 5amino acids amino acid linear peptide unknown 30 Gln Phe Glu Phe Lys 1 511 amino acids amino acid linear peptide unknown Other 1...1 where Xaaat position 1 is Met, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu 31 XaaGly Thr Leu Gly Ile Val Cys Pro Ile Cys 1 5 10 14 amino acids amino acidlinear peptide unknown 32 Met Gly Thr Leu Gly Ile Val Cys Pro Ile CysSer Gln Lys 1 5 10 11 amino acids amino acid linear peptide unknown 33Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys 1 5 10

What is claimed is:
 1. A method of making a polypeptide, which methodcomprises maintaining a cell containing a plasmid comprising a codingsequence coding for expression of a polypeptide under conditionspermitting expression of said polypeptide, the polypeptide comprising afirst peptide and a second peptide linked by a peptide bond, the firstpeptide being a peptide which controls intracellular trafficking of apeptide to which it is attached, and the second peptide consisting of asequence 12-18 amino acids in length comprising the sequence Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:16).
 2. A method ofmaking a polypeptide, which method comprises maintaining a cellcontaining a plasmid comprising a coding sequence coding for expressionof a polypeptide under conditions permitting expression of saidpolypeptide, the polypeptide comprising a first peptide and a secondpeptide linked by a peptide bond, the first peptide being a peptidewhich controls intracellular trafficking of a peptide to which it isattached, and the second peptide consisting of a sequence 8-18 aminoacids in length comprising the sequence Thr Leu Gly Ile Val Cys Pro Ile(SEQ ID NO:20).
 3. A method of inducing an immune response in a mammal,which method comprises administering to a mammal a nucleic acidcomprising a coding sequence coding for expression of a polypeptidecomprising a first peptide and a second peptide linked by a peptidebond, the first peptide being a peptide which controls intracellulartrafficking of a peptide to which it is attached, and the second peptideconsisting of a sequence 12-18 amino acids in length comprising thesequence Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:20).4. A method of inducing an immune response in a mammal, which methodcomprises administering to a mammal a nucleic acid comprising a codingsequence coding for expression of a polypeptide comprising a firstpeptide and a second peptide linked by a peptide bond, the first peptidebeing a peptide which controls intracellular trafficking of a peptide towhich it is attached, and the second peptide consisting of a sequence8-18 amino acids in length comprising the sequence Thr Leu Gly Ile ValCys Pro Ile (SEQ ID NO:20).
 5. A method of inducing an immune responsein a mammal, which method comprises administering to a mammal a plasmidcomprising a coding sequence coding for expression of a polypeptidecomprising a first peptide and a second peptide linked by a peptidebond, the first peptide being a peptide which controls intracellulartrafficking of a peptide to which it is attached, and the second peptideconsisting of a sequence 12-18 amino acids in length comprising thesequence Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:16).6. A method of inducing an immune response in a mammal, which methodcomprises administering to a mammal a plasmid comprising a codingsequence coding for expression of a polypeptide comprising a firstpeptide and a second peptide linked by a peptide bond, the first peptidebeing a peptide which controls intracellular trafficking of a peptide towhich it is attached, and the second peptide consisting of a sequence8-18 amino acids in length comprising the sequence Thr Leu Gly Ile ValCys Pro Ile (SEQ ID NO:20).
 7. A method of inducing an immune responsein a mammal, which method comprises administering to a mammal amicroparticle comprising (a) a polymeric matrix; and (b) a plasmidcomprising a coding sequence coding for expression of a polypeptidecomprising a first peptide and a second peptide linked by a peptidebond, the first peptide being a peptide which controls intracellulartrafficking of a peptide to which it is attached, and the second peptideconsisting of a sequence 12-18 amino acids in length comprising thesequence Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:16).8. The method of claim 7, wherein the mammal is a human.
 9. The methodof claim 8, wherein the human suffers from, or is at risk of a conditionselected from the group consisting of exophytic condyloma, flatcondyloma, cervical cancer, respiratory papilloma, conjunctivalpapilloma, genital-tract HPV infection, and cervical dysplasia.
 10. Amethod of inducing an immune response in a mammal, which methodcomprises administering to a mammal a microparticle comprising (a) apolymeric matrix; and (b) a plasmid comprising a coding sequence codingfor expression of a polypeptide comprising a first peptide and a secondpeptide linked by a particle bond, the first peptide being a peptidewhich controls intracellular trafficking of a peptide to which it isattached, and the second peptide consisting of a sequence 8-18 aminoacids in length comprising the sequence Thr Leu Gly Ile Val Cys Pro Ile(SEQ ID NO:20).
 11. The method of claim 10, wherein the mammal is ahuman.
 12. The method of claim 11, wherein the human suffers from, or isat risk of, a condition selected from the group consisting of exophyticcondyloma, flat condyloma, cervical cancer, respiratory papilloma,conjunctival papilloma, genital-tract HPV infection, and cervicaldysplasia.
 13. A method of inducing a cell mediated, anti-HPV immuneresponse in a mammal, which method comprises administering to the mammala DNA comprising the sequence of SEQ ID NO:7.
 14. A method of inducingan immune response in a patient, which method comprises administering tothe patient a microparticle having a diameter of less than 20 micronsand consisting essentially of a polymeric matrix and a nucleic acidmolecule, wherein the polymeric matrix consists essentially of PLGA andthe nucleic acid molecule comprises the sequence of SEQ ID NO:7.
 15. Amethod of inducing an immune response in an mammal, which methodcomprises administering a nucleic acid comprising a coding sequencecoding for expression of a peptide less than 19 amino acids in length,wherein the peptide comprises the amino acid sequence Leu Met Gly ThrLeu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:16).
 16. The method of claim15, wherein the peptide's amino acid sequence comprises Leu Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:3).
 17. The method ofclaim 15, wherein the peptide's amino acid sequence comprises Xaa LeuMet Gly Thr Leu Gly Ile Val Cys Pro Ile Cys, Xaa being Met, Ala, Ser,Arg, Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:19).
 18. The method of claim17, wherein Xaa is Ala or Met.
 19. The method of claim 15, wherein thepeptide's amino acid sequence comprises Leu Leu Met Gly Thr Leu Gly IleVal Cys Pro Ile Cys Ser Gln Lys (SEQ ID NO:25).
 20. A method of inducingan immune response in an mammal, which method comprises administering anucleic acid comprising a coding sequence coding for expression of apeptide less than 19 amino acids in length, wherein the peptidecomprises the amino acid sequence Gly Thr Leu Gly Ile Val Cys Pro Ile(SEQ ID NO:21).
 21. The method of claim 20, wherein the peptide's aminoacid sequence comprises Xaa Gly Thr Leu Gly Ile Val Cys Pro Ile Cys, Xaabeing Met, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:31). 22.The method of claim 20, wherein the peptide's amino acid sequencecomprises Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:33).23. The method of claim 21, wherein the peptide's amino acid sequenceconsists of Xaa Gly Thr Leu Gly Ile Val Cys Pro Ile Cys, Xaa being Met,Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:31).
 24. The methodof claim 22, wherein the peptide's amino acid sequence consists of MetGly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys (SEQ ID NO:32). 25.The method of claim 1, wherein the sequence of the first peptidecomprises the amino acid sequence Met Ala Ile Ser Gly Val Pro Val LeuGly Phe Phe Ile Ile Ala Val Leu Met Ser Ala Gln Glu Ser Trp Ala (SEQ IDNO: 18).
 26. The method of claim 1, wherein the amino acid sequence ofthe second peptide is Xaa Leu Met Gly Thr Leu Gly Ile Val Cys Pro IleCys, Xaa being Met, Leu, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu (SEQID NO:19).
 27. The method of claim 1, wherein the amino acid sequence ofthe second peptide is Ala Leu Met Gly Thr Leu Gly Ile Val Cys Pro IleCys (SEQ ID NO:4).
 28. The method of claim 25, wherein the amino acidsequence of the second peptide is Xaa Leu Met Gly Thr Leu Gly Ile ValCys Pro Ile Cys, Xaa being Met, Leu, Ala, Ser, Arg, Lys, Gly, Gln, Asp,or Glu (SEQ ID NO:19).
 29. The method of claim 25, wherein the aminoacid sequence of the second peptide is Ala Leu Met Gly Thr Leu Gly IleVal Cys Pro Ile Cys (SEQ ID NO:4).
 30. The method of claim 3, whereinthe sequence of the rirst peptide comprises the amino acid sequence MetAla Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val Leu Met SerAla Gln Glu Ser Trp Ala (SEQ ID NO: 18).
 31. The method of claim 3,wherein the amino acid sequence of the second peptide is Xaa Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys, Xaa being Met, Leu, Ala, Ser, Arg,Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:19).
 32. The method of claim 3,wherein the amino acid sequence of the second peptide is Ala Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:4).
 33. The method ofclaim 30, wherein the amino acid sequence of the second peptide is XaaLeu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys, Xaa being Met, Leu,Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:19).
 34. The methodof claim 30, wherein the amino acid sequence of the second peptide isAla Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:4). 35.The method of claim 5, wherein the sequence of the first peptidecomprises the amino acid sequence Met Ala Ile Ser Gly Val Pro Val LeuGly Phe Phe Ile Ile Ala Val Leu Met Ser Ala Gln Glu Ser Trp Ala (SEQ IDNO: 18).
 36. The method of claim 5, wherein the amino acid sequence ofthe second peptide is Xaa Leu Met Gly Thr Leu Gly Ile Val Cys Pro IleCys, Xaa being Met, Leu, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu (SEQID NO:19).
 37. The method of claim 5, wherein the amino acid sequence ofthe second peptide is Ala Leu Met Gly Thr Leu Gly Ile Val Cys Pro IleCys (SEQ ID NO:4).
 38. The method of claim 35, wherein the amino acidsequence of the second peptide is Xaa Leu Met Gly Thr Leu Gly Ile ValCys Pro Ile Cys, Xaa being Met, Leu, Ala, Ser, Arg, Lys, Gly, Gln, Asp,or Glu (SEQ ID NO:19).
 39. The method of claim 35, wherein the aminoacid sequence of the second peptide is Ala Leu Met Gly Thr Leu Gly IleVal Cys Pro Ile Cys (SEQ ID NO:4).
 40. The method of claim 7, whereinthe polymeric matrix consists essentially of PLGA.
 41. The method ofclaim 7, wherein rhe microparticle has a diameter of 0.02 to 20 microns.42. The method of claim 7, wherein the microparticle has a diameter ofless than about 11 microns.
 43. The method of claim 7, wherein thesequence of the first peptide comprises the amino acid sequence Met AlaIle Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val Leu Met Ser AlaGln Glu Ser Trp Ala (SEQ ID NO: 18).
 44. The method of claim 7, whereinthe amino acid sequence of the second peptide is Xaa Leu Met Gly Thr LeuGly Ile Val Cys Pro Ile Cys, Xaa being Met, Leu, Ala, Ser, Arg, Lys,Gly, Gln, Asp, or Glu (SEQ ID NO:19).
 45. The method of claim 7, whereinthe amino acid sequence of the second peptide is Ala Leu Met Gly Thr LeuGly Ile Val Cys Pro Ile Cys (SEQ ID No:4).
 46. The method of claim 43,wherein the amino acid sequence of the second peptide is Xaa Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys, Xaa being Met, Leu, Ala, Ser, Arg,Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:19).
 47. The method of claim 43,wherein the amino acid sequence of the second peptide is Ala Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:4).
 48. The method ofclaim 40, wherein the sequence of the first peptide comprises the aminoacid sequence Met Ala Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile IleAla Val Leu Met Ser Ala Gln Glu Ser Trp Ala (SEQ ID NO: 18).
 49. Themethod of claim 40, wherein the amino acid sequence of the secondpeptide is Xaa Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys, Xaabeing Met, Leu, Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu (SEQ IDNO:19).
 50. The method of claim 40, wherein the amino acid sequence ofthe second peptide is Ala Leu Met Gly Thr Leu Gly Ile Val Cys Pro IleCys (SEQ ID NO:4).
 51. The method of claim 48, wherein the amino acidsequence of the second peptide is Xaa Leu Met Gly Thr Leu Gly Ile ValCys Pro Ile Cys, Xaa being Met, Leu, Ala, Ser, Arg, Lys, Gly, Gln, Asp,or Glu (SEQ ID NO:19).
 52. The method of claim 48, wherein the aminoacid sequence of the second peptide is Ala Leu Met Gly Thr Leu Gly IleVal Cys Pro Ile Cys (SEQ ID NO:4).
 53. The method of claim 42, whereinthe sequence of the first peptide comprises the amino acid sequence MetAla Ile Ser Gly Val Pro Val Leu Gly Phe Phe Ile Ile Ala Val Leu Met SerAla Gln Glu Ser Trp Ala (SEQ ID NO: 18).
 54. The method of claim 42,wherein the amino acid sequence of the second peptide is Xaa Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys, xaa being Met, Leu, Ala, Ser, Arg,Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:19).
 55. The method of claim 42,wherein the amino acid sequence of the second peptide is Ala Leu Met GlyThr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:4).
 56. The method ofclaim 53, wherein the amino acid sequence of the second peptide is XaaLeu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys, Xaa being Met, Letu,Ala, Ser, Arg, Lys, Gly, Gln, Asp, or Glu (SEQ ID NO:19).
 57. The methodof claim 53, wherein the amino acid sequence of the second peptide isAla Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys (SEQ ID NO:4). 58.The method of claim 10, wherein the polymeric matrix consistsessentially of PLGA.
 59. The method of claim 10, wherein themicroparticle has a diameter of 0.02 to 20 microns.
 60. The method ofclaim 10, wherein the microparticle has a diameter of less than about 11microns.
 61. The method of claim 1, wherein the cell is a mammalian Bcell or APC.
 62. The method of claim 25, wherein the cell is a mammalianB cell or APC.
 63. The method of claim 26, wherein the cell is amammalian B cell or APC.
 64. The method of claim 27, wherein the cell isa mammalian B cell or APC.
 65. The method of claim 3, wherein the mammalis a human.
 66. The method of claim 4, wherein the mammal is a human.67. The method of claim 5, wherein the mammal is a human.
 68. The methodof claim 6, wherein the mammal is a human.
 69. The method of claim 65,wherein the human suffers from, or is at risk of a condition selectedfrom the group consisting of exophytic condyloma, flat condyloma,cervical cancer, respiratory papilloma, conjunctival papilloma,genital-tract HPV infection, and cervical dysplasia.
 70. The method ofclaim 66, wherein the human suffers from, or is at risk of a conditionselected from the group consisting of exophytic condyloma, flatcondyloma, cervical cancer, respiratory papilloma, conjunctivalpapilloma, genital-tract HPV infection, and cervical dysplasia.
 71. Themethod of claim 67, wherein the human suffers from, or is at risk of acondition selected from the group consisting of exophytic condyloma,flat condyloma, cervical cancer, respiratory papilloma, conjunctivalpapilloma, genital-tract HPV infection, and cervical.
 72. The method ofclaim 68, wherein the human suffers from, or is at risk of a conditionselected from the group consisting of exophytic condyloma, flatcondyloma, cervical cancer, respiratory papilloma, conjunctivalpapilloma, genital-tract HPV infection, and cervical dysplasia.
 73. Amethod of inducing a cell-mediated, anti-HPV immune response in amammal, which method comprises administering to the mammal a DNAcomprising the sequence of SEQ ID NO:5.
 74. A method of inducing animmune response in a patient, which method comprises administering tothe patient a microparticle having a diameter of less than 20 micronsand consisting essentially of a polymeric matrix and a nucleic acidmolecule, wherein the polymeric matrix consists essentially of PLGA andthe nucleic acid molecule comprises the sequence of SEQ ID NO:5.
 75. Amethod of inducing a cell-mediated, anti-HPV immune response in amammal, which method comprises administering to the mammal a DNAcomprising the sequence of nucleotides 3290-3413 of SEQ ID NO:7.
 76. Amethod of inducing an immune response in a patient, which methodcomprises administering to the patient a microparticle having a diameterof less than 20 microns and consisting essentially of a polymeric matrixand a nucleic acid molecule, wherein the polymeric matrix consistsessentially of PLGA and the nucleic acid molecule comprises the sequencenucleotides 3290-3413 of SEQ ID NO:7.
 77. The method of claim 75,wherein the DNA comprises the sequence of nucleotides 3219-3624 of SEQID NO:7.
 78. The method of claim 77, wherein the nucleic acid moleculecomprises the sequence of nucleotides 3219-3624 of SEQ ID NO:7.
 79. Themethod claim 73, wherein the mammal is a human.
 80. The method claim 75,wherein the mammal is a human.
 81. The method claim 77, wherein themammal is a human.